The aim of the proposed research is to elucidate the quaternary structure and mechanism of action of the enzyme hydrogenase. Having isolated pure hydrogenase, we propose to investigate the nature of the active site, its reaction with substrate and inhibitors, the arrangement of the protein subunits and iron-sulfur center in the active enzyme, and the mechanism by which the enzyme activates hydrogen. We will determine the number of electrons accepted by the enzyme from the substrate and the mechanism by which the enzyme hydride intermediate is formed. Experiments are outlined to demonstrate conclusively the existence of an enzyme hydride. The reaction of the active site with the inhibitors, O2, CO, and NO will be studied by absorption, CD, and EPR spectra to determine the state of the enzyme they react with and the mechanism by which the inhibition takes place. We will determine how the two subunits and the iron-sulfur active site are arranged in the active hydrogenase. This will involve isolation and separation of the subunits under different conditions and determining whether they are identical and possess catalytic activity. The composition of the subunits and their spectral properties will establish the quaternary structure.